The present invention concerns novel pharmaceutical compositions. In particular, the pharmaceutical compositions of the invention comprise cysteine protease modulators. The pharmaceutical compositions of the invention are preferably used for the treatment of viral infections, and diseases resulting from inappropriate apoptosis.
Cysteine proteases are a major family of peptide-bond-cleaving hydrolases, defined as proteases in which the thiol group of a cysteinee residue serves as nucleophile in a catalysis. All known cysteinee peptidases require a second residuexe2x80x94an adjacent histidinexe2x80x94for catalysis. While the role of the histidine has been postulated to be a general base in analogy to well-understood serine proteases, it has been clearly demonstrated in theoretical studies that the catalytic histidine cannot act as a base, rather that it acts by donating a proton to the substrate. Cysteinee proteases have been found in the previous literature in viruses, bacterial protozoa, plants, mammals and fungi.
There are currently known 38 families of cysteinee proteases (C1-C38), most of which are divided into 5 separately evolved clans (CA-CE). Clan CB enzymes are chymotrypsin-like cysteine proteases containing a His/Cys diad (catalytic histidine preceding catalytic cysteinee in the linear sequence), and responsible for proteolytic cleavage of pol polyproteins (containing the RNA polymerase). These enzymes, commonly hydrolyse glutaminyl bonds, and act on crucial cell proteins as additional substrates.
Peptidases of Clan CA include vital mammalian enzymes such as papain or cathepsins. The normal activity of these enzymes is essential and their activity should not be inhibited by any type of pharmaceutical composition.
Clan CC includes sixteen (16) families of papain-like viral peptidases (C6-C9,C16,C21,C23,C27-29,C31-C36), comprising a cys/his diad. Despite sequences similar to Clan CA enzymes, these viral proteins are functionally similar to Clan CB enzymes, which cleave viral polyproteins.
Clan CD is represented by a single family (C14), which comprises cytosolic endopeptidases found only in animals. Cytosolic endopeptidases are involved in the process of apoptosis (programmed cell death).
There is no structural data regarding peptidases of Clan CE (family C5 adenovirus endopeptidase), as well as untyped enzymes. One untyped family, however, C13, which includes medically important proteases such as Schistosoma mansoni haeomoglobinase, is similar to the substrate specificity of Clan CB enzymes (asparaginyl compared to glutaminyl bonds). Additionally, C13 has a low sensitivity to E64. Interestingly, this latter property may indicate a fold similar to Clan CB chymotrypsin-like-enzymes.
Picornaviruses are single-stranded positive RNA viruses that are encapsulated in a protein capsid. These viruses cause a wide range of diseases in man and animal including common cold, poliomyelitis, hepatitis A, encephalitis, meningitis and foot-and-mouth disease, as well as diseases in plants such as the potty disease in potatoes. After inclusion into the host cell, the picornaviral RNA is translated into a 247-kDa protein that is co- and post-translationally cleaved yielding eleven (11) mature proteins. Cysteine proteases denoted 2A and 3C, which are part of the picornaviral self polyprotein are responsible for these cleavages. The 2A protease cleaves co-translationally between the structural and non-structural proteins and the 3C protease cleaves post-translationally the remaining cleavage sites except one.
Having been recognized as important proteins in the maturation of the picornaviral life cycle the 3C and 2A proteases have been a prime target for extensive structural and mechanistic investigations during the last few years. Recently, their mechanism and structural features have been determined (Kreisberg et al, Organic Reactivity: Physical and Biological Aspects, 110-122 (1995)).
Site-directed mutagenesis studies (Cheah K. C. et al, J. Biol. Chem., 265 (13):7187-7189 (1990)) confirmed by X-ray studies (Matthews et al, Cell, 77:761-771, (1994)) led to the finding that the catalytic site of 3C is composed of the following amino acids: Cys in position 146, Glu/Asp in position 71 and His in position 40. These three amino acids in the catalytic site of the 3C enzyme constitute a hybrid between the amino acids at the catalytic site of cysteine proteases and serine proteases.
The 3C protease has been shown by mutagenesis and crystallography to depend on a his/cys diad (His40, Cys146xe2x80x94rhinovirus numbering). A third conserved residue in the 3C protease, Asp 71, was initially considered analogous to Asn175 (the third member in the catalytic triad of papain), however crystallography has shown this residue to be of minor catalytic importance.
Due to the involvement of various cysteine proteases in many disorders and diseases ranging from microorganism infection (viral and bacterial) to inflammatory and tumor processes, there have been recently many attempts to find inhibitors for cysteine proteases (Otto and Schimeister, Chem. Rev., 97:133-171, 1997)).
There have also been attempts to find suitable inhibitors of the picornavirus 3C and 2A proteases in order to treat viral infections. By inhibiting these proteases, the production of new virions can be avoided because there are no native cellular proteases that can replace the cleavage activity of the viral proteases. Therefore, finding an efficient inhibitor against 3C and/or 2A picornavirus proteases will lead to the production of an anti-viral pharmaceutical composition against a large number of viral diseases occurring both in man and in animal.
The first agent found as an inhibitor of the 3C protease is Thysanone, an antibiotic compound obtained from Thysanophora peniciloides (Singh et al, Tetrahedron Lett., 32:5279-82 (1991)). However, this compound was not developed into a pharmaceutical composition because it was found to be an efficient inhibitor of the enzyme elastase present in erythrocytes.
Two additional antibiotic compounds of fungal origin, citrinin hydrate and radicinin, were obtained by screening microbial extracts (Kadam et al, J. Antibiotics 7:836-839 (1994)). These novel two compounds showed a lower level of inhibition than thysanone. The same year a new compound termed kalafungin, which is also an antibiotic compound, was discovered by structural comparison to radicinin. Kalafungin was found to be a better inhibitor (by three orders of magnitude) than radicinin and citrinin hydrate (McCall et al, Biotechology, 12:1012-1016 (1994)).
Another group of inhibitors, substituted isatins, has also been examined (S. E. Webber, et al., Med. Chem., 39:5072-5082, 1996). Certain members of this group show significant inhibition of 3C proteases with concentrations in the nanomolar range, but are highly toxic. Other members of the group are relatively non-toxic, but have poor antiviral activity. It has recently been shown that peptidyl Michael acceptors inhibit rhinovirus replication at low micromolar concentrations with a therapeutic index exceeding ten (10) (Kong et al., J. Med. Chem., 41:2579-2587 (1998). Rhinovirus inhibition has also been accomplished at nanomolar concentrations at peptidyl Michael acceptors (Dragovich et al., J. Med. Chem., 41:2819-2834 (1998)). Thus, none of the above inhibitors has been demonstrated to be clinically useful possessing a sufficiently high therapeutic index with favorable toxicology and bioavailability profiles.
Transition-state analogs are well established as enzyme and protease inhibitors (Barrett, A. J. and Salvesen, G., Proteinase Inhibitors, Elsevier, 1986). Functional groups such as ketone, aldehyde, chloromethyl-ketone (REVS) and recently isatin are widely used for the inhibition of serine and cysteine proteases. Class-specificity is achieved by utilization of phosphine or boron geometries (serine proteases) or groups such as epoxide (Albeck, M., Fluss, S. and Persky, R., J. Am. Chem. Soc., 118:3591-3596, 1996), cyclopropenone (Ando, R. and Morinaka, Y., J. Am. Chem. Soc., 115:1174-1175, 1993) and vinyl-sulfones (Bromme, D., et al., Biochem, J., 315:85-89, 1996).
This approach, protease inhibition through transition-state mimicry, yields highly potent inhibitors when combined with target-specific amino-acid residues or their peptidomimetic equivalent. Unfortunately, the high molecular weight and complexity of potent TS-analogs frequently cause transport problems, which result in diminished in vivo efficacy.
In other diseases it is desired to activate cysteine proteases. These diseases are characterized by deficient apoptosis, i.e. by insufficient programmed cell death. These diseases include certain types of cancer, viral diseases and certain autoimmune diseases.
One of the key apoptotic elements identified is Apopain (caspase-3) (Nicholson, Nature Biotech., 14:297-301 (1996)). Modulators of this protein are sought for the modulation of apoptosis and the provision of novel therapeutics, Inhibitors for Apopain are useful for the treatment of diseases in which excessive apoptosis occurs, including neurodegenerative diseases such as Alzheimer, Parkinson and Huntington and cardiovascular diseases such as ischemic cardiac damage. Enhancers of this protein are useful for the treatment of diseases in which insufficient apoptosis occurs, such as cancer, viral infections and certain autoimmune diseases.
Compounds such as those discussed in WPI abstract 021538, JP abstract 03271261, EP application 0244363, DE application 4126543, FR application 2482859, and certain references in the Merck Index have been identified to treat certain diseases discussed above. However, such compounds do not treat such diseases by reacting with certain 3C protease or 3C protease-like proteins, which are essential to viral replication and the activity of various proteins. Thus, it would be highly desirable to construct protease modulators in particular, cysteine protease modulators that can be administered for various pharmaceutical and medicinal purposes to a subject.
The present invention concerns modulators of cysteine proteases and more specifically modulators of picornavirus 3C-cysteine protease and of similar proteins.
The present invention is based on the finding that several chemical compounds are capable of inhibiting the picornavirus 3C-cysteine protease.
Thus, according to the first aspect of the invention termed xe2x80x9cthe inhibiting aspectxe2x80x9d there are provided inhibitors of picornavirus 3C proteases and inhibitors of proteins having similar activity.
The present invention is based on further findings discovered by x-ray analysis that there exists a structural similarity between Apopain and the rhinovirus 3C protease. Similarity in active site and catalytic machinery between the two enzymes has suggested similar mechanism and activity. Thus, compounds which enhance or inhibit 3C protease are assumed to have activity also towards Apopain.
Thus by a second aspect termed xe2x80x9cthe enhancing aspectxe2x80x9d, the present invention concerns enhancers of 3C-like proteases such as Apopain.
In one aspect, the invention is a method for the modulation of a cysteine protease target comprising exposing the target to a chemical composition having a core structure 
wherein
A and Axe2x80x2 together form a C6 aromatic or C5-C7 aliphatic ring, and R1 is hydrogen or a hydrocarbon moiety of 1 to 10 carbons which is optionally substituted, wherein the target has an active site and catalytic mechanism similar to apopain and rhinovirus 3C protease
In another aspect, the invention is a method for the modulation of a cysteine protease target comprising exposing the target to a chemical composition having an orthohydroxy keto aryl core structure 
wherein
R1 is hydrogen, a hydrocarbon moiety of 1 to 10 carbons optionally substituted with an aryl, an amino optionally substituted with C1-C2 arylalkyl or C8 alkyl, an aryl optionally substituted with hydroxyl or keto, methoxy, C2 arylalkyl optionally substituted with hydroxyl;
T1, T2, T3, and T4 are selected from the group consisting of C, O, N or S;
Zxe2x80x2 is hydrogen, hydroxyl, C1-C4 alkoxy or xe2x80x94OCH2CONH2;
Y is hydrogen, halogen, hydroxyl, nitro, cyano, methyl or xe2x80x94COOCH2CH3 with the proviso that when R1 is a lower alkyl or arylalkyl, Zxe2x80x2 is hydrogen or C1-C4 alkoxy and T1, T2, T3, and T4 are all C, Y cannot be hydrogen or alkyl;
Yxe2x80x2 is hydrogen, halogen, hydroxyl, nitro, methyl or methoxy;
R3 is hydrogen, hydroxyl, methyl or C1-C3 alkoxy;
alternatively R1 together with Zxe2x80x2, Yxe2x80x2 together with R3, R3 together with Y, or Zxe2x80x2 together with Yxe2x80x2 can form an aromatic or aliphatic ring structure optionally heterocyclic, optionally substituted with hydroxyl, keto, C1-C4 alkanoyl, carboxyl, alkyloxycarbonyl, or phenyl optionally substituted with hydroxyl,
wherein the target has an active site and catalytic mechanism similar to apopain and rhinovirus 3C protease.
In another aspect, the invention is a method for the modulation of a cysteine protease target comprising exposing the target to a chemical composition having an orthohydroxy keto aryl core structure 
wherein
R1 is selected from the group consisting of:
(i) hydrogen or a hydrocarbon chain from 1 to about 10 carbons long selected from the group consisting of saturated, unsaturated and fluorinated, wherein the hydrocarbon chain is unsubstituted or substituted with at least one R11, wherein R11 is selected from the group consisting of:
(ia) C1-C4 alkyl, C2-C4 alkenyl, C3-C8 cycloalkyl, C1-C3 alkoxy or aryl which may be unsubstituted or substituted with halogen, hydroxy, methyl, ethyl, acetyl, carboxamide, nitro, sulfamide, phenyl or sulfamyl;
(ib) halogen, cyano, nitro, amino, hydroxy, adamantyl, carbamyl, carbamyloxy or keto;
(ic) an oligopeptide of 1-4 amino acid residues, and
(id) NR13R14, CO2R13, O(Cxe2x95x90OR13), SO2R13, SOR13R14, (Cxe2x95x90O)NR13R14, or NR14(Cxe2x95x90O)R13;
wherein:
R13 is selected from the group consisting of hydrogen, phenyl, benzyl, C1-C6 alkyl and C3-C6 cycloalkyl; and
R14 is selected from the group consisting of hydrogen, hydroxyl, and benzyl;
(ii) an oligopeptide of 1 to 5 amino acids;
(iii) C3-C6 cycloalkyl, C6-C10 bicycloalkyl, C3-C7 cycloalkylmethyl, or C7-C10 arylalkyl, which may be additionally substituted with R11 as defined above; and
(iv) C1-C5 alkoxy optionally substituted with 1-3 R11, NH-W or NW2, wherein W is a substituent as defined in (i), (ii) or (iii) above;
T1, T2, T3, and T4 are selected from the group consisting of C, O, N or S;
Zxe2x80x2 is hydrogen, hydroxyl or C1-C4 alkoxy optionally containing 1-2 unsaturations and substituted with 1-3 R15), or C5-C7 carbocyclic or heterocyclic ring system connected to R1 optionally containing 1-2 unsaturations, wherein R15 is selected from phenyl optionally substituted with 1-3 R14, naphthyl optionally substituted with 1-3 R14, or a C3-C6 heterocyclic ring system fused to an aromatic ring optionally containing 1-2 nonbenzenoid unsaturations and optionally substituted with 1-3 R14;
Y and Yxe2x80x2 are independently selected from the group consisting of:
(i) hydrogen, hydroxyl, halogen, C1-C4 haloalkyl, C1-C4 haloalkoxy; or C1-C3 alkyl which may be additionally substituted with 1-3 R11 as defined above, with the proviso that when R1 is a lower alkyl or arylalkyl, Zxe2x80x2 is hydrogen or C1-C4 alkoxy and T1, T2, T3, and T4 are all C, Y cannot be hydrogen or alkyl; and
(ii) carbamyl, cyano, vinyl, nitro, sulfamyl, or sulfamido; and
R3 is selected from the group consisting of hydrogen, hydroxyl, methyl, C1-C3 hydrocarbon chain or C1-C3 alkoxy, allyl and amino;
alternatively R1 together with Zxe2x80x2, Yxe2x80x2 together with R3, R3 together with Y, or Zxe2x80x2 together with Yxe2x80x2 can form an aromatic or aliphatic ring structure, optionally heterocyclic, optionally substituted with 1-4 R11, 
wherein the target has an active site and catalytic mechanism similar to apopain and rhinovirus 3C protease.
Preferred chemical compositions for these methods of the present invention have the following orthohydroxy keto aryl core structure 
wherein Zxe2x80x2 is hydrogen, hydroxyl, methoxy, or xe2x80x94OCH2CONH2; Y is hydrogen, halogen nitro, cyano, methyl, or xe2x80x94COOCH2CH3 with the proviso that when Zxe2x80x2 is hydrogen or methoxy, Y cannot be hydrogen; Yxe2x80x2 is hydrogen, halogen, hydroxyl, methyl or methoxy; and R3 is hydrogen, hydroxyl, methyl or methoxy. Exemplary compositions having this orthohydroxy keto aryl core structure include compositions wherein: (1) Zxe2x80x2 and R3 are hydrogen and Y and Yxe2x80x2 are Cl or Br; (2) Zxe2x80x2 and R3 are hydrogen, Y is nitro, and Yxe2x80x2 is Cl or methyl; (3) Zxe2x80x2, Yxe2x80x2 and R3 are hydrogen and Y is cyano; (4) Zxe2x80x2 and R3 are hydroxyl, Y is hydrogen or xe2x80x94COOCH2CH3, and Yxe2x80x2 is hydrogen; (5) Zxe2x80x2 is hydroxyl, R3 is hydrogen, and Y and Yxe2x80x2 are Cl; and (6) Zxe2x80x2 is xe2x80x94OCH2CONH2, and R3, and Yxe2x80x2 are hydrogen.
Other preferred chemical compositions for these methods of the present invention have the following orthohydroxy keto aryl core structure 
wherein Zxe2x80x2 is hydrogen, hydroxyl, C1-C4 alkoxy or xe2x80x94OCH2CONH2; Y is hydrogen, halogen, hydroxyl, nitro, cyano, methyl or xe2x80x94COOCH2CH3 with the proviso that when Zxe2x80x2 is hydrogen or C1-C4 alkoxy, Y cannot be hydrogen or methyl; Yxe2x80x2 is hydrogen, halogen, hydroxyl, nitro, methyl or methoxy; and R3 is hydrogen, hydroxyl, methyl or C1-C3 alkoxy.
Other preferred chemical compositions for these methods of the present invention have the following orthohydroxy keto aryl core structure 
wherein Zxe2x80x2 is hydrogen, hydroxyl, methoxy or xe2x80x94OCH2CONH2; Yxe2x80x2 is hydrogen or halogen; R3 is hydroxyl or methoxy; and Y is hydrogen, halogen or hydroxyl with the proviso that when Zxe2x80x2 is hydrogen, Y cannot be hydrogen. Exemplary compositions having this orthohydroxy keto aryl core structure include compositions wherein: (1) Zxe2x80x2 is hydroxyl, R3 is methoxy, and Yxe2x80x2 and Y are hydrogen or Cl; (2) Zxe2x80x2 is methoxy, R3 is hydroxyl, Yxe2x80x2 is hydrogen or Cl, and Y is hydroxyl or Cl; (3) Zxe2x80x2 is hydrogen, R3 is hydroxyl, and Yxe2x80x2 is hydrogen or Cl and Y is Cl; and (4) Zxe2x80x2 is xe2x80x94OCH2CONH2, R3 is methoxy, and Yxe2x80x2 and Y are Cl.
Other preferred chemical compositions for these methods of the present invention have the following orthohydroxy keto aryl core structure 
wherein Zxe2x80x2 is hydrogen or hydroxyl; Yxe2x80x2 is hydrogen, nitro, Cl or I; R3 is hydrogen or hydroxyl; and Y is hydrogen, Cl or I. Exemplary compositions having this orthohydroxy keto aryl core structure include compositions wherein: (1) Zxe2x80x2 and R3 are hydroxyl, and Yxe2x80x2 and Y are hydrogen; (2) Zxe2x80x2 and R3 are hydrogen, and Yxe2x80x2 and Y are Cl or I; and (3) Zxe2x80x2, R3 and Y are hydrogen and Yxe2x80x2 is nitro.
Other preferred chemical compositions for these methods of the present invention have the following orthohydroxy keto aryl core structure 
wherein Zxe2x80x2 and R3 are hydrogen; and W is CH2-phenyl, CH2CH2-phenyl or (CH2)7CH3.
Other preferred chemical compositions for these methods of the present invention have the following orthohydroxy keto aryl core structure 
wherein R3 and Y are hydrogen.
Other preferred chemical compositions for these methods of the present invention have the following orthohydroxy keto aryl core structure 
wherein Zxe2x80x2, Yxe2x80x2 and Y are hydrogen and R3 is methoxy.
Other preferred chemical compositions for these methods of the present invention have the following orthohydroxy keto aryl core structure 
wherein Zxe2x80x2 is hydrogen; Y is hydrogen; Yxe2x80x2 is hydrogen, methyl, or halogen; and R3 is hydrogen or methyl. Exemplary compositions having this orthohydroxy keto aryl core structure include compositions wherein: (1) Zxe2x80x2, R3 and Y are hydrogen and Yxe2x80x2 is hydrogen, methyl, Cl or Br; and (2) Zxe2x80x2 and Y are hydrogen, R3 is methyl, and Yxe2x80x2 is Cl.
Other preferred chemical compositions for these methods of the present invention have the following orthohydroxy keto aryl core structure 
wherein Yxe2x80x2, R3, and Y are hydrogen.
Other preferred chemical compositions for these methods of the present invention have the following orthohydroxy keto aryl core structure 
wherein Zxe2x80x2 is OH, Yxe2x80x2 and Y are H, and R3 is methyl.
Other preferred chemical compositions for these methods of the present invention have the following orthohydroxy keto aryl core structure 
wherein Zxe2x80x2 is hydrogen or hydroxyl; Y is halogen; Yxe2x80x2 is hydrogen or halogen; R3 is hydrogen or hydroxyl; and R11 is hydrogen or hydroxyl. Exemplary compositions having this orthohydroxy keto aryl core structure include compositions wherein: (1) Zxe2x80x2 is hydrogen, R3, and R11 are hydrogen or hydroxyl, Yxe2x80x2 is hydrogen or Cl, and Y is Cl; (2) Zxe2x80x2 is hydroxyl, R3 and R11 are hydrogen, and Yxe2x80x2 and Y are hydrogen or Cl; and (3) Zxe2x80x2, R3 and R11 are hydroxyl, and Yxe2x80x2 and Y are hydrogen.
Other preferred chemical compositions for these methods of the present invention have the following orthohydroxy keto aryl core structure 
wherein Y is hydrogen or Cl; Yxe2x80x2 is hydrogen or Cl; R3 is hydrogen or hydroxyl; R11 is hydrogen or hydroxyl; and R12 is hydrogen or hydroxyl. Exemplary compositions having this orthohydroxy keto aryl core structure include compositions wherein: (1) R3, R11 and R12 are hydrogen or hydroxyl, and Yxe2x80x2 and Y are hydrogen or Cl; (2) R3 is hydroxyl, Yxe2x80x2 and Y are hydrogen, and R11 and R12 are hydrogen or hydroxyl; and (3) R3 is hydroxyl, Yxe2x80x2 and Y are Cl, and R11 and R12 are hydrogen.
In another aspect, the invention is a composition having the following structure 
wherein R1 is hydrogen, a hydrocarbon chain from 1 to about 10 carbons optionally substituted with an aryl, C1-C3 alkoxy, amino optionally substituted with C1-C10 hydrocarbon or C1-C3 arylalkyl, aryl optionally substituted with hydroxyl or keto, or arylalkyl optionally substituted with hydroxyl; R3 is hydrogen, hydroxyl, methyl, or C1-C3 alkoxy; Yxe2x80x2 is hydrogen, hydroxyl, halogen, nitro, cyano, C1-C3 alkyl, or C1-C3 alkoxy; and Y is hydrogen, hydroxyl, halogen, nitro, cyano, or COOCH2CH3. Exemplary compositions having this orthohydroxy keto aryl core structure include compositions wherein: (1) R1 is methyl, and Yxe2x80x2, R3 and Y are hydrogen, thereby comprising 2-(2-acetyl-3-hydroxyphenoxy)-acetamide; and (2) R1 is (CH2)4CH3, R3 is methoxy, and Y and Yxe2x80x2 are Cl.
The term xe2x80x9camino acidxe2x80x9d used above is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including for example hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine. Furthermore, the term xe2x80x9camino acidxe2x80x9d includes both D- and L-amino acids. The term xe2x80x9coligopeptidexe2x80x9d refers to a series of amino acids linked by peptide bonds.
Suitable sequences of amino acids can be chosen according to the teachings of Cordingley et al., J. Biol Chem., 265(16):9062-9066 (1990). Additionally, various amino acids may be screened as follows: (1) a desirable protease, such as the 3C picornavirus protease is immobilized to a solid support, (2) candidate sequences are brought into contact with the immobilized protease, (3) residues that bind to the imnmobilized proteases are chosen as candidate sequences.
The pharmaceutical compositions of the present invention are suitable for the treatment of diseases manifested by the activity of cysteine proteases of the CB Clan (families C3, C4, C24, C30, C37 and C38) of the CD Clan (family C14), of the CE clan and of family C13.
The term xe2x80x9cdiseases, which manifestation is dependent on the activity cysteine proteasesxe2x80x9d, refers to a disease that can be treated, prevented, alleviated or cured by inhibition of cysteine proteases of the CB Clan, the CD Clan, the CE clan and the C13 family. Preferably, the inhibition is of xe2x80x9cpicornavirus 3C-like cysteine proteasesxe2x80x9d, which are cysteine proteases having an active site similar to the active site of the 3C protease, (a catalytic dyad of Histidine and Cysteine) and most preferably of 3C-cysteine proteases.
Thus, most preferably, the pharmaceutical compositions of the invention according to the inhibition aspect of the invention are for the treatment of viral infections and of diseases wherein excessive apoptosis is implicated and wherein apoptosis should be reduced most preferably for picornaviral infection, neurodegenerative disease and certain cardiovascular diseases.
The pharmaceutical compositions of the invention according to the inhibition aspect of the invention are suitable for the treatment of common colds, allergic rhinitis, poliomyelitis, hepatitis-A, encephalitis, meningitis, hand-foot-and-mouth disease, encephalomyocarditis, summer flu (enteroviral upper respiratory infection), asthma, various allergies, myocarditis, acute hemorrhagic conjunctivitis, disseminated neonatal infection and Borhnolm""s disease. All the above are diseases which manifestation is dependent on the activity of a cysteine protease of the CB clan.
The inhibitors of the pharmaceutical compositions of the present invention selectively bind to the picornaviral proteases, essentially in a similar manner as the viral coded natural substrate of the proteases, and compete with the substrates for proteases. This competition serves to inhibit viral maturation and thus to inhibit disease progression in vivo.
The pharmaceutical compositions of the present invention are suitable also for the treatment of diseases manifested by the activity of the cysteine proteases of the CD clan, i.e apoptosis-involved diseases, which includes activation, as in cancer, as well as inhibition (as in neurodegenerative diseases) of apoptosis.
The pharmaceutical compositions of the present invention are also suitable for the treatment of adenovirus-involved diseases.
Thus the present invention, according to its inhibition aspect, further provides a method for treatment of viral infection, in particular a picornaviral infection by administrating to a subject in need of such treatment a pharmaceutically acceptable amount of a compound of formulae (I) to (VI), which has protease inhibitor activity optionally together with a pharmaceutically acceptable carrier.
The present invention further concerns, according to its inhibition aspect, a method for the treatment of cardiovascular diseases such as ischemic cardiac damage by administering to a subject, in need of such treatment, a pharmaceutically acceptable amount of a compound of formulae (I) to (VI), which has protease inhibitor activity optionally together with a pharmaceutically acceptable carrier.
Further, the present invention, according to its inhibition aspect, provides a method for the treatment of neurodegenerative diseases such as Alzheimer""s, Parkinson""s and Huntington""s, a pharmaceutically acceptable amount of a compound of the formulae (I) to (VI), which has protease inhibitor activity optionally together with a pharmaceutically acceptable carrier.
As will no doubt be appreciated by the person skilled in the art, the above Formulae I-VI cover a large number of possible compounds, some of which are inhibitors and some are enhancers, and from those which are inhibitors some are more effective inhibitors of cysteine proteases of the above types than others
In order to determine which of the compounds are suitable as 3C protease inhibitors, according to the inhibitory aspect of the present invention, compounds may be screened for inhibitory activities according to one of the following assays:
Assays for screening picornaviral protease inhibitors
I. Birch et al. (Protein Expression and Purification, 6:609-618 (1995)) have developed a continuous fluorescence assay to determine kinetic parameters and to screen potential HRV14 3C protease inhibitors. The assay consists of a consensus peptide for rhinoviruses connected to a fluorescence donor group (anthranilic acid, Anc) at the N terminal and to an acceptor group (p-NO2-Phe; Pnp) at the P4 position, both groups flanking the scissile bond (Gln/Gly). The substrate peptide consists of the following sequence: Anc-Thr-Leu-Phe-Gln-Gly-Pro-Val-Pnp-Lys. There is a linear time dependent increase in fluorescence intensity as the substrate is cleaved, which allows continuous monitoring of the reaction. Multiwell plates containing one inhibitor per well allows for rapid screening by measuring the fluorescence intensity in each well.
II. Heinz et al. (Antimicrobial Agents and Chemotherapy, 267-270 (1996)) developed an assay method for measuring 3C protease activity and inhibition using the substrate biotin-Arg-Ala-Glu-Leu-Gln-Gly-Pro-Tyr-Asp-Glu-Lys-fluorescein-isothiocyanate. Cleavage mixtures containing inhibitors are allowed to bind to avidin beads and are subsequently washed. The resultant fluorescence of the bead is proportional to the degree of inhibition.
III. Another assay developed by McCall et al. (Bio/Technology, 12 1012-1016 (1994)) measures in addition to the inhibitory effects of the candidate inhibitors, their capability to enter into cells so that a high capacity screen for compounds inhibiting the 3C protease of HRV-1B is developed. The assay uses a recombinant strain of E-coli expressing both the protease and a tetracycline resistance gene modified to contain the minimal 3C protease cleavage sequence. Cultures growing in microtiter plates containing tetracycline are treated with potential inhibitors. Culture with no inhibition of the 3C protease, show reduced growth due to cleavage of the essential gene product. Normal growth is seen only in cultures that contains an effective 3C protease inhibitor.
IV. An assay was developed in our lab based on a protein consisting of the 3C protease fused to DHFR. The cleavage of the fusion protein by external 3C protease (type 1A) is monitored by gel-electrophoresis. The degree of cleavage is proportional to the ratio of low molecular weight proteins (3C and DHFR) to intact fusion protein, as observed on the gel.
V. Other assays developed for inhibition of other cysteine proteases are well known in the art.
The pharmaceutical compositions of the invention according to the xe2x80x9cenhancement aspectxe2x80x9d are suitable for diseases manifested by deficient apoptosis, i.e. inappropriate activity of Apopain and of other xe2x80x9c3C protease-like proteinsxe2x80x9d. The term xe2x80x9c3C protease-like proteinsxe2x80x9d refers to cysteine proteases with active site structures similar to that of a picornavirus 3C protease as discovered by homology or by x-ray analysis. An example of such a protease is Apopain. In particular, the diseases are characterized by insufficient apoptosis and include among others autoimmune diseases, viral-caused infectious diseases and certain types of cancer.
The diseases may be treated, prevented, alleviated or cured by compositions of the invention having Apopain enhancing activity.
Diseases in which insufficient apoptosis is implicated will be cured by Apopain enhancement leading to normal or excessive levels of apoptosisxe2x80x94thus, for example, certain cancers originating from subnormal levels of programmed cell death will be eliminated following the restoration of normal levels of apoptosis or the establishment of higher than normal levels of apoptosis.
The present invention further concerns a method for treatment of autoimmune diseases, viral-caused infectious diseases and certain types of cancer as well as cardiovascular diseases such as ischemic cardiac damage by administering to a subject in need of such treatment a pharmaceutically acceptable array of a compound of formulae I-VI, which has protease enhancing activity
As will be no doubt appreciated by a person skilled in the art, formulae I-VI above cover a large number of possible compounds, some of which are inhibitors and some are enhancers, and from those which are enhancers some are more effective than others.
In order to determine compounds that are most suitable as enhancers, compounds may be screened by the following additional assay (VI)
VI: Apopain (Caspase-3) Regulation Assay
The FluorAce(trademark) Apopain Assay Kit (Bio-rad) was employed in multiwell format. Compounds assayed were diluted 5-fold in distilled water (from stock solutions in ethanol or DMSO) and centrifuged (5xe2x80x2, 14K rpm). 10 xcexcL of the supernatant was further diluted in wells containing 100 xcexcL distilled water and 40 xcexcL 6xc3x97Buffer (41.7 mM PIPES, pH 7.4, 8.3 mM EDTA, 0.42% CHAPS, 20.8 mM DTT). This process was carried out twice to yield duplicate wells for each compound. Control wells were prepared by addition of EtOH and DMSO (5-fold diluted in distilled water) into wells containing 6xc3x97Buffer and distilled water (40 xcexcL and 100 xcexcL, respectively), to form 8 wells with each solvent. Enzyme (stock solution 10-fold diluted in distilled water, see Bio-rad booklet 4100119) was added to one set of compound-containing cells and to 8 control wells (4 with ethanol and 4 with DMSO). The plate was preincubated for xcx9c80xe2x80x2 at 27xc2x0-28xc2x0 C. Substrate (Z-DEVD-AFC, 490 xcexcM, 40 xcexcL) was then added to all wells. The plate was left at room temperature and fluorescence (360/40↑530/20↓) was measured at several time points (FL500 fluorimetric reader, Bio-tek instruments). Fluorescence at enzyme-containing wells was background-subtracted at each time point and initial rates were determined by linear regression (typically R2 greater than 0.97). Percentage inhibition and activation was determined by relating the slope with compound to the control slope (with appropriate solvent). IC50 values are extrapolated.
Pharmaceutically acceptable carriers are well known in the art and are disclosed, for instance, in Sprowl""s American Pharmacy, Dittert, L. (ed.), J. B. Lippincott Co., Philadelphia, 1974, and Remington""s Pharmaceutical Sciences, Gennaro, A. (ed.), Mack Publishing Co., Easton, Pa., 1985.
Pharmaceutical compositions of the compounds of the present invention, or of pharmaceutically acceptable salts thereof, may be formulated as solutions or lyophilized powders for parenteral administration. Powders may be reconstituted by addition of a suitable diluent or other pharmaceutically acceptable carrier prior to use. The liquid formulation is generally a buffered, isotonic, aqueous solution, but a lipophilic carrier, such as propylene glycol optionally with an alcohol, can be more appropriate for compounds of this invention. Examples of suitable diluents are normal isotonic saline solution, standard 5% dextrose in water of buffered sodium or ammonium acetate solution. Such a formulation is especially suitable for parenteral administration, but can also be used for oral administration or contained in a metered dose inhaler of nebulizer for insufflation. It may be desirable to add excipients such as ethanol, polyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia, polyethylene glycol, mannitol, sodium chloride or sodium citrate
Alternately, the compounds of the invention may be encapsulated, tableted or prepared in an emulsion or syrup for oral administration. Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate preparation of the composition. Liquid carriers include syrup, soy bean oil, peanut oil, olive oil, glycerin, saline, ethanol, and water. Solubizing agents, such as dimethylsulfoxide, ethanol or formamide, may also be added. Carriers, such as oils, optionally with solubizing excipients, are especially suitable. Oils include any natural or synthetic non-ionic water-immiscible liquid, or low melting solid capable of dissolving lipophilic compounds. Natural oils, such as triglycerides are representative. In fact, another aspect of this invention is a pharmaceutical composition comprising a compound of formula (I) and an oil.
Solid carriers include starch, lactose, calcium sulfate dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin. Solubilizing agents, such as dimethylsulfoxide or formamide, may also be added. The carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax. The pharmaceutical preparations are made following the conventional techniques of pharmacy involving milling, mixing, granulating, and compressing for tablet forms; or milling, mixing and filling for hard gelatin capsule forms. When a liquid carrier is used, the preparation will be in the form of a syrup, elixir, emulsion or an aqueous or non-aqueous suspension. Such a liquid formulation can be administered directly p.o. or filled into a soft gelatin capsule.
For rectal administration, a pulverized powder of the compounds of this invention may be combined with excipients such as cocoa butter, glycerin, gelatin or polyethylene glycols and molded into a suppository. The pulverized posers may also be compounded with an oily preparation, gel, cream or emulsion, buffered or unbuffered, and administered through a transdermal patch.
Nasal administration of the compounds of the invention can also be used especially for the treatment of common cold and allergic rhinivity.
The present invention also concerns a method for the detection of picornaviral infection. According to a method of the invention, a compound of the invention bearing a detectable label (for example attached to one of its substituents) is incubated with a sample suspected of containing picornaviruses, under conditions enabling binding of the compound to proteases. Preferably, the sample should be treated with a lysing agent in order to release the picornavirus proteins from inclusion bodies. Then it is determined whether the labeled compounds of the invention are bound to any proteins in assay. A positive answer (beyond a predetermined control level) is indicative of the presence of a picornavirus in the assayed sample.
The present invention further concerns several novel chemical compounds denoted in the examples as follows:
SA#121, SA#132, SA#116, SA#118, SA#134, SA#135, SA#120, SA#127, SA#128, SA#15*, SA#16*, SA#107*, SA#108*, SA#110**, SA#43, SA#109*, SA#139, SA#51.
previously described
commercially available
The invention will now be described in reference to some non-limiting examples.